A biodegradable hydrogel and its preparation method

Abstract

In its first aspect, the present invention provides a biodegradable hydrogel and a method for preparing the same. The biodegradable hydrogel comprises components A and B: Component A comprises the following: NHS-PEG-NHS, halide-PEG-halide, and solvent A is water or saline solution; Component B comprises the following: a macromolecular substance containing an amino group, and solvent B is water or saline solution. This method utilizes a reaction between succinimide groups and amino groups to form a gel, instantly fixing wounds in situ. Subsequently, a slow reaction between halogen elements and tertiary amines forms a gel, enhancing the gel's modulus while producing quaternary ammonium groups with antibacterial properties, thus improving the system's antibacterial efficacy. Furthermore, the gel exhibits hemostatic, self-healing, and tissue repair properties.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical wound repair materials and relates to an injectable biodegradable hydrogel with the characteristics of automatic enhancement of in-situ modulus and antibacterial effect and a preparation method thereof. Background Art

[0002] Hydrogels, with their high water content and close resemblance to human tissue, are high-quality materials for wound repair scaffolds. Injectable hydrogels, due to their excellent fluidity before gel formation, can perfectly fill irregular injury gaps and fully cover the damaged area when injected into the injury site. Subsequently, under certain conditions, they can instantly gel at the wound site, achieving the purpose of fixing the wound and providing in situ treatment. In general, this method offers advantages in application, such as low cost, convenient operation, minimal invasiveness, and low risk of bacterial infection. It has been widely studied and applied in the medical field in recent years. For example, CN113461973A discloses a medical hydrogel that is cross-linked from aldehyde-terminated star-shaped multi-arm polyethylene glycol, polyethyleneimine, and hydrazide-terminated multi-arm polyethylene glycol, resulting in a fast-gelling and long-term stable injectable hydrogel. Another example is CN105963792A, which discloses a Michael addition-based antibacterial and biodegradable medical hydrogel with the excellent properties of fast gelling and low swelling. However, most injectable gels suffer from a single function and a modulus that is not compatible with the implantation environment. In the design of implants, it has become a trend to introduce multifunctional auxiliary devices to promote wound repair and modulus adaptation to the environment.

[0003] The environment inside the human body is complex and diverse, and it is under frequent mechanical movement for a long time. In actual situations, suddenly implanting an implant with too high a modulus at the wound site will make the human body feel uncomfortable. Although soft implants will make the human body more accepting, the modulus of the soft object in the later stage of implantation cannot support the defect cavity, resulting in reduced healing efficiency. This problem can be solved if the modulus of the implant can adapt to the in vivo environment over time. At the same time, bacterial infection at the wound site is one of the important factors that hinder wound healing. If measures are not taken to assist the wound in resisting the invasion of external bacteria, there is a probability of infection by various bacteria, causing varying degrees of inflammation and delaying the natural healing process. Therefore, it is necessary to design and synthesize an injectable hydrogel with a modulus that is compatible with the in vivo environment and has multiple functions such as antibacterial and hemostatic.

[0004] Previously, we proposed a hydrogel dressing that can quickly gel and slowly strengthen after injection (Yang Jintao, Yuan Jingfeng, Zheng Siyu, Zhang Dong, A method for preparing a hydrogel dressing that can quickly gel and slowly strengthen after injection, CN202110636898.1). This work uses a soft and fast Schiff base network and a strong and slow epoxy amino cross-linked network to construct a slowly self-reinforcing injectable gel system, but the system is still weak in resisting bacterial invasion in practical applications. In view of this, the present invention replaces the epoxy amino cross-linked gel network with a quaternary ammonium bond cross-linked network that is slowly generated by tertiary amine halogens. The modulus of this gel system continues to improve the antibacterial effect while adapting to the in vivo environment. It is a more innovative injectable gel with in situ enhanced mechanical and antibacterial properties. Summary of the Invention

[0005] To enhance patient comfort during implant placement and meet the modulus requirements for repairing gaps in implant-supported tissue later in the process, this paper proposes a method for preparing an injectable biodegradable hydrogel with in situ self-enhancing mechanical and antimicrobial properties. In addition to rapid gelation and gradual modulus enhancement, this hydrogel also exhibits enhanced antimicrobial properties.

[0006] To achieve the above strategy, the technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a biodegradable hydrogel, wherein the biodegradable hydrogel is made of component A and component B:

[0008] The component A comprises the following components: NHS-PEG-NHS, halide-PEG-halide, and solvent A; the solvent A is water or saline solution;

[0009] The number average molecular weight of NHS-PEG-NHS is 400-5000 Da; the number average molecular weight of halide-PEG-halide is 400-5000 Da (preferably 2000-2100 Da);

[0010] The component B comprises the following components: a macromolecular substance containing an amino group, a solvent B; the solvent B is water or a saline solution;

[0011] The amino-containing macromolecular substance is one or a mixture of two or more of tertiary amino chitosan (CS), tertiary amine polylysine (EPL), gelatin, and polyethyleneimine (PEI) (preferably tertiary amino chitosan or polyethyleneimine, particularly preferably polyethyleneimine, and most preferably polyethyleneimine with a number average molecular weight of 10 kDa);

[0012] The mass ratio of the NHS-PEG-NHS, halide-PEG-halide and amino-containing macromolecular substance is 100:20-90:15-180 (preferably 100:60:100); the mass of the NHS-PEG-NHS is 25-100 mg / mL (preferably 50 mg / mL) based on the total volume of component A and component B.

[0013] Furthermore, the component A is composed of the following components in the following mass fractions: NHS-PEG-NHS, halide-PEG-halide, and solvent A; solvent A is water or saline solution;

[0014] The component B is composed of the following components in mass fractions: a macromolecular substance containing an amino group and a solvent B; the solvent B is water or a saline solution.

[0015] In an embodiment of the present invention, the structure of the NHS-PEG-NHS is one of the following:

[0016] II III

[0017] In formula III, x is an integer between 1 and 10. The present invention recommends that x be 3 or 6.

[0018] In an embodiment of the present invention, NHS-PEG-NHS is prepared from PEG and a disuccinimidyl ester in an organic solvent under the action of a catalyst. The disuccinimidyl ester is one or a mixture of two or more of N,N'-disuccinimidyl carbonate (DSC), disuccinimidyl suberate (DSS), or disuccinimidyl glutarate (DSG), preferably N,N'-disuccinimidyl carbonate. The catalyst is one or a mixture of two or more of triethylamine, pyridine, and 4-dimethylaminopyridine (DMAP), preferably 4-dimethylaminopyridine. Other substances with similar structures can be synthesized by reference.

[0019] Specifically, the NHS-PEG-NHS was prepared as follows:

[0020] PEG is dissolved in organic solvent A to obtain a PEG solution; a substance containing succinimide groups at both ends is dissolved in organic solvent B to obtain solution B; the PEG solution and solution B are mixed evenly, a catalyst is added, mixed evenly, and stirred at room temperature for 6 to 12 hours (10 hours in the embodiment of the present invention). The resulting reaction solution is post-treated to obtain the NHS-PEG-NHS.

[0021] Furthermore, the post-treatment is as follows: rotary evaporating the reaction solution, washing the obtained solid with ether, filtering, and drying the obtained filter cake to obtain the NHS-PEG-NHS.

[0022] Furthermore, the molar ratio of the PEG, the substance containing succinimide groups at both ends, and the catalyst is 1:0.5-5:0.3-2 (preferably 1:2:0.8).

[0023] Furthermore, the organic solvent A and the organic solvent B are each independently one of acetonitrile, dichloromethane, and chloroform, or a mixture of two or more thereof.

[0024] Furthermore, the volume of the organic solvent A is 0.75 to 15 mL / g (preferably 1.5 to 15 mL / g) based on the mass of the PEG; the volume of the organic solvent B is 3 to 15 mL / g based on the mass of the substance containing succinimide groups at both ends.

[0025] Commonly used halide-PEG-halide in the art is Cl-PEG-Cl or Br-PEG-Br.

[0026] In an embodiment of the present invention, the structure of the halide-PEG-halide is as follows:

[0027] I

[0028] In formula I, R1 is a halogenated C1-C6 alkyl group or , wherein R2 is a halogenated C1-C6 alkyl group.

[0029] In an embodiment of the present invention, in formula I, R1 is 2-chloroethyl, 2-bromoethyl or , wherein R2 is 4-chloromethyl.

[0030] In an embodiment of the present invention, a method for preparing a halide-PEG-halide of Formula I is provided. The halide-PEG-halide of Formula I is prepared from PEG (polyethylene glycol) and a substance containing halogens at both ends in an organic solvent under the action of a catalyst. The substance containing halogens at both ends is one or a mixture of two or more of bis(2-chloroethyl)amine, di(2-bromoethyl)amine, and 4-(chloromethyl)benzoyl chloride (preferably 4-(chloromethyl)benzoyl chloride). The catalyst is one or a mixture of two or more of triethylamine, pyridine, and 4-dimethylaminopyridine, preferably triethylamine. Other polymers with similar structures can be synthesized similarly.

[0031] Specifically, the halide-PEG-halide represented by formula I is prepared as follows:

[0032] PEG is dissolved in organic solvent C to obtain a PEG solution, a catalyst is added, and the mixture is uniformly mixed; a substance with halogen groups at both ends is dissolved in organic solvent D to obtain a solution D; the solution D is injected into an ice bath for 5 to 10 minutes. o C., injected into the PEG solution for mixing, and stirred at room temperature for 6 to 24 hours (12 hours in the embodiment of the present invention). The resulting reaction solution was post-treated to obtain the halide-PEG-halide represented by formula I.

[0033] Furthermore, the post-treatment is as follows: the reaction solution is filtered and then rotary evaporated, the obtained solid is washed with deionized water, the supernatant is collected after centrifugation, and the obtained liquid is freeze-dried to obtain the halide-PEG-halide represented by formula I.

[0034] Furthermore, the molar ratio of the PEG, the substance with halogen at both ends, and the catalyst is 1:1-5:1-5 (preferably 1:2:2).

[0035] Furthermore, the organic solvent C and the organic solvent D are each independently one of dichloromethane, chloroform, and acetone, or a mixture of two or more thereof, and in the embodiment of the present invention, dichloromethane.

[0036] Furthermore, the volume of the organic solvent A is 1 to 10 ml / g based on the mass of the PEG; the volume of the organic solvent B is 2 to 10 ml / g based on the mass of the substance with halogen at both ends.

[0037] Furthermore, the solvent A and solvent B are each independently one of water, PBS, borate buffer solution, and physiological saline, or a mixture of two or more thereof. In the embodiment of the present invention, the solvent A and solvent B are PBS with a pH of 7.4.

[0038] Furthermore, the present invention recommends that the amino-containing macromolecular substance have a number average molecular weight of 5 to 100 kDa (5 to 10 kDa in the present embodiment). Experiments have shown that amino-containing macromolecular substances below 100 kDa do not affect gel network formation.

[0039] In an embodiment of the present invention, the tertiary amine-modified amino macromolecule is prepared from an amino macromolecule, an amine compound, and an organic solvent; the amino macromolecule is one or a mixture of two or more of chitosan and polylysine; the amine compound is one or a mixture of two of N,N-dimethylaminochloropropane hydrochloride and N,N-dimethyl-oxiranylmethylamine, preferably N,N-dimethylaminochloropropane hydrochloride.

[0040] Furthermore, the tertiary amine chitosan (CS) and tertiary amine polylysine of the present invention are prepared according to the following method: chitosan or polylysine is dissolved in an organic solvent E, an alkaline substance is added to alkalize for 1 hour, an amine compound is added and refluxed at room temperature for 5 hours, and the resulting reaction solution is post-treated to obtain the tertiary amine chitosan (CS) or tertiary amine polylysine.

[0041] Furthermore, the post-treatment is as follows: the reaction solution is filtered, the filter cake is washed with acetone (twice), deionized water is added, the pH is adjusted to 5 with acetic acid, and the solution is dialyzed with deionized water for 5 days. The retentate is freeze-dried to obtain the tertiary amine chitosan (CS) or tertiary amine polylysine.

[0042] Furthermore, the molar ratio of the chitosan or polylysine to the amine compound is 1:15 to 100 (preferably 1:60).

[0043] Furthermore, the organic solvent E is one or a mixture of two or more of isopropyl alcohol, dichloromethane, and chloroform.

[0044] Furthermore, the alkaline substance is one or a mixture of two or more of NaOH, NaHCO3, and Na2CO3, and the mass ratio of the alkaline substance to the amino macromolecule is 1:1 to 2.

[0045] Furthermore, the volume of the organic solvent E is 10-20 ml / g based on the mass of the chitosan or polylysine.

[0046] In a second aspect, the present invention provides a method for preparing the above-mentioned biodegradable hydrogel, which comprises: mixing the component A and the component B to obtain the biodegradable hydrogel.

[0047] In one embodiment of the present invention, the method comprises: separately filling Component A and Component B into different barrels of a double-barreled syringe, mixing and extruding, to obtain the biodegradable hydrogel. This is the injectable biodegradable hydrogel with in situ mechanical and antibacterial self-reinforcement characteristics. Furthermore, preferably, Component A and Component B have the same volume.

[0048] Preferably, the temperature for injection molding is 25-40°C.

[0049] Compared with the prior art, the beneficial effects of the present invention are mainly:

[0050] 1. Material selection: Most of the raw materials are natural and non-toxic materials, which have good biocompatibility and hydrophilicity while maintaining excellent biodegradability.

[0051] 2. Operation: It is applied by injecting the gel into the form of a double-barreled syringe, which is simple to operate and low in cost.

[0052] 3. Functionality: Primarily, the present invention utilizes a rapid reaction between succinimide groups and amino groups to form a gel, instantly fixing wounds in situ. The subsequent slow reaction between halogen elements and tertiary amines enhances the gel modulus while simultaneously producing quaternary ammonium groups with antimicrobial properties, enhancing the system's antimicrobial efficacy. This injectable hydrogel exhibits significant antimicrobial efficacy that strengthens over time. In particular, hydrogels prepared with halogen-terminated polyethylene glycol (PEG) exhibit significantly better enhancement than those produced with halogen-containing small molecules. Furthermore, the gel exhibits hemostatic, self-healing, and tissue repair properties.

[0053] 4. Application: The present invention has a wide range of medical applications, such as internal and external surgical bleeding blockage, in vivo tissue defect repair, in vivo and in vitro drug release therapy, and in vivo and in vitro inflammation treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 Injectability of the injectable gel in Example 1.

[0055] Figure 2 The mixture of Comparative Example 7 did not form a gel.

[0056] Figure 3 ac are the self-reinforcement properties of the single / double cross-linked network injectable gels in Examples / Comparative Examples 1, 2, and 6, respectively.

[0057] Figure 4 Antibacterial properties of the injectable gels of Example 1 (double network) and Comparative Example 1 (single network).

[0058] Figure 5 Sterilization rates of the injectable gels in Examples 1-6 and Comparative Examples 1-6. DETAILED DESCRIPTION

[0059] The present invention is further described below by means of specific examples, but the protection scope of the present invention is not limited thereto.

[0060] Example 1 Preparation of double network gel

[0061] Thoroughly dissolve 10g of PEG (0.005mol, number-average molecular weight 2000Da) in 15ml of dichloromethane. Simultaneously, dissolve 2.56g of DSC (0.01mol) in 15ml of acetonitrile by sonication for 15 minutes. Combine the two solutions, add 0.5g of DMAP (0.004mol), and sonicate again for 30 minutes. Stir the suspension at 25°C for 10 hours. The product is rotary evaporated, washed three times with ether, and filtered. The filter cake is dried in a vacuum oven at 40°C for later use to obtain NHS-PEG-NHS (number-average molecular weight 2282Da).

[0062] Dissolve 20g PEG (0.01mol) in 60ml dichloromethane, add 2g triethylamine (0.02mol), and mix well; at the same time, dissolve 3.78g 4-(chloromethyl)benzoyl chloride (0.02mol) in 10ml dichloromethane, and inject the mixture in an ice bath for 5-10 min. o The mixture was injected into the PEG solution at 40 °C and stirred at room temperature for 12 h. The resulting product was filtered and the filtrate was evaporated. The remaining solid was washed with deionized water and the supernatant was centrifuged and freeze-dried for later use to obtain Cl-PEG-Cl (number average molecular weight 2305 Da).

[0063] Dissolve 100 mg of NHS-PEG-NHS and 60 mg of Cl-PEG-Cl in 1 ml of PBS (1.45 mmol / mL sodium chloride, 0.09 mmol / mL sodium hydrogen phosphate, 0.005 mmol / mL sodium dihydrogen phosphate, pH 7.4), denoted as component A. Dissolve 100 mg of PEI (number-average molecular weight 10 kDa, the same below), denoted as component B, in 1 ml of PBS. Components A and B are separately placed in a double-barreled syringe and injected at the site of action to rapidly form a gel.

[0064] Example 2 Preparation of double network gel

[0065] Thoroughly dissolve 20 g of PEG (0.005 mol, number-average molecular weight 4000 Da) in 30 ml of chloroform. Simultaneously, dissolve 2.56 g of DSC (0.01 mol) in 15 ml of acetonitrile by sonication for 15 minutes. Combine the two solutions, add 0.5 mL of pyridine (0.006 mol), and sonicate again for 30 minutes. Stir the suspension at 25°C for 10 hours. Rotary evaporate the product, wash it three times with ether, and filter it. The filter cake is dried in a vacuum oven at 40°C until ready for use. This yields NHS-PEG-NHS (number-average molecular weight 4282 Da).

[0066] Dissolve 40g PEG (0.01mol) in 120ml dichloromethane, add 2g triethylamine (0.02mol), and mix well; at the same time, dissolve 4.6g di(2-bromoethyl)amine (0.02mol) in 10ml dichloromethane, and inject into ice bath for 5-10min. o The mixture was injected into the PEG solution at 40 °C and stirred at room temperature for 12 h. The resulting product was filtered and then rotary evaporated. The remaining solid was washed with deionized water and the supernatant was centrifuged and freeze-dried for later use to obtain Br-PEG-Br (number average molecular weight 4140 Da).

[0067] Dissolve 5g of chitosan (0.005 mol, number-average molecular weight 10 kDa, same below) in 100ml of isopropanol and stir at 60°C for 5 minutes. Add 5g of NaOH to alkalize for 1 hour, then add 5g of N,N-dimethylaminopropane chloride hydrochloride (0.03 mol) and reflux for 5 hours. Filter the solution, wash the remaining solid twice with acetone, add deionized water, adjust the pH to 5 with acetic acid, dialyze against deionized water for 5 days, and lyophilize to obtain tertiary amine chitosan.

[0068] Dissolve 150 mg of NHS-PEG-NHS and 40 mg of Br-PEG-Br in 1 ml of PBS (1.45 mmol / mL sodium chloride, 0.09 mmol / mL sodium hydrogen phosphate, 0.005 mmol / mL sodium dihydrogen phosphate, pH 7.4), denoted as component A. Dissolve 100 mg of tertiary amine chitosan in 1 ml of PBS, denoted as component B. Components A and B are separately placed in a double-barreled syringe and injected at the site of action to rapidly form a gel.

[0069] Example 3 Preparation of double network gel

[0070] Thoroughly dissolve 2 g of PEG (0.005 mol, number-average molecular weight 400 Da) in 15 ml of acetonitrile. Simultaneously, sonicate 3.68 g of DSS (0.01 mol) in 15 ml of acetonitrile for 30 minutes. Combine the two solutions, add 0.7 mL of triethylamine (0.005 mol), and sonicate again for 30 minutes. Stir the suspension at 25°C for 10 hours. Rotary evaporate the product, wash it three times with ether, and filter it. Dry the filter cake in a vacuum oven at 40°C to obtain NHS-PEG-NHS (number-average molecular weight 850 Da).

[0071] Dissolve 4 g PEG (0.01 mol) in 30 ml dichloromethane, add 2.44 g DMAP (0.02 mol), and mix well. At the same time, dissolve 3.57 g bis(2-chloroethyl)amine (0.02 mol) in 10 ml dichloromethane, and inject the mixture in an ice bath for 5 to 10 minutes. o The mixture was injected into the PEG solution at 60°C and stirred at room temperature for 12 hours. The resulting product was filtered and rotary evaporated. The remaining solid was washed with deionized water, centrifuged, and the supernatant was lyophilized for later use to obtain Cl-PEG-Cl (number-average molecular weight 540 Da). 3g of chitosan was dissolved in 100ml of isopropanol and stirred at 60°C for 5 minutes. 5g of NaOH was added to alkalize the solution for 1 hour, followed by 5g of N,N-dimethylaminopropane chloride hydrochloride, and the reaction was refluxed for 5 hours. The remaining solid was filtered and washed twice with acetone. After adding deionized water, the pH was adjusted to 5 with acetic acid, dialyzed against deionized water for 5 days, and lyophilized to obtain tertiary amine chitosan.

[0072] Dissolve 150 mg of NHS-PEG-NHS and 40 mg of Cl-PEG-Cl in 1 ml of PBS (1.45 mmol / mL sodium chloride, 0.09 mmol / mL sodium hydrogen phosphate, 0.005 mmol / mL sodium dihydrogen phosphate, pH 7.4), denoted as component A. Dissolve 100 mg of tertiary amine chitosan in 1 ml of PBS, denoted as component B. Components A and B are separately placed in a double-barreled syringe and injected at the site of action to rapidly form a gel.

[0073] Example 4 Preparation of double network gel

[0074] Thoroughly dissolve 10 g of PEG (0.01 mol, number-average molecular weight 1000 Da) in 15 ml of acetonitrile. Simultaneously, dissolve 3.26 g of DSG (0.01 mol) in 15 ml of chloroform by sonication for 15 minutes. Combine the two solutions, add 0.5 mL of pyridine (0.006 mol), and sonicate again for 30 minutes. Stir the suspension at 25°C for 10 hours. The product is then rotary evaporated, washed three times with ether, and filtered. The filter cake is dried in a vacuum oven at 40°C to yield NHS-PEG-NHS (number-average molecular weight 1270 Da).

[0075] Dissolve 10 g of PEG (0.01 mol) in 30 ml of dichloromethane, add 1.6 mL of pyridine (0.02 mol), and mix well. At the same time, dissolve 4.6 g of di(2-bromoethyl)amine (0.02 mol) in 10 ml of dichloromethane, and inject the mixture in an ice bath for 5 to 10 minutes. o The mixture was injected into the PEG solution at 40 °C and stirred at room temperature for 12 h. The resulting product was filtered and then rotary evaporated. The remaining solid was washed with deionized water and the supernatant was centrifuged and freeze-dried for later use to obtain Cl-PEG-Cl (number average molecular weight 1140 Da).

[0076] Dissolve 100 mg of NHS-PEG-NHS and 60 mg of Cl-PEG-Cl in 1 ml of PBS (1.45 mmol / mL sodium chloride, 0.09 mmol / mL sodium hydrogen phosphate, 0.005 mmol / mL sodium dihydrogen phosphate, pH 7.4), denoted as component A. Dissolve 80 mg of PEI in 1 ml of PBS, denoted as component B. Components A and B are separately placed in a double-barreled syringe and injected at the site of action to rapidly form a gel.

[0077] Example 5 Preparation of double network gel

[0078] Thoroughly dissolve 10g of PEG (0.005 mol, number-average molecular weight 2000 Da) in 15ml of dichloromethane. Simultaneously, sonicate 2.56g of DSC (0.01 mol) in 15ml of acetonitrile for 15 minutes. Combine the two solutions, add 0.5g of DMAP (0.004 mol), and sonicate again for 30 minutes. Stir the suspension at 25°C for 10 hours. Rotary evaporate the product, wash it three times with ether, and filter it. The filter cake is dried in a vacuum oven at 40°C until ready for use, yielding NHS-PEG-NHS (number-average molecular weight 2282 Da).

[0079] Dissolve 20g PEG (0.01mol) in 60ml dichloromethane, add 2g triethylamine (0.02mol), and mix well; at the same time, dissolve 3.78g 4-(chloromethyl)benzoyl chloride (0.02mol) in 10ml dichloromethane, and inject the mixture in an ice bath for 5-10 min. o The mixture was injected into the PEG solution at 40 °C and stirred at room temperature for 12 h. The resulting product was filtered and then rotary evaporated. The remaining solid was washed with deionized water and the supernatant was centrifuged and freeze-dried for later use to obtain Cl-PEG-Cl (number average molecular weight 2140 Da).

[0080] Dissolve 100 mg of NHS-PEG-NHS and 60 mg of bis(2-chloroethyl)amine in 1 ml of water (Component A). Dissolve 100 mg of gelatin (number average molecular weight 15 kDa, the same below) in 1 ml of water (Component B). Place components A and B separately into a double-barreled syringe and inject at the site of action to rapidly form a gel.

[0081] Example 6 Preparation of double network gel

[0082] Thoroughly dissolve 10g of PEG (0.005 mol, number-average molecular weight 2000 Da) in 15ml of dichloromethane. Simultaneously, sonicate 2.56g of DSC (0.01 mol) in 15ml of acetonitrile for 15 minutes. Combine the two solutions, add 0.5g of DMAP (0.004 mol), and sonicate again for 30 minutes. Stir the suspension at 25°C for 10 hours. Rotary evaporate the product, wash it three times with ether, and filter it. The filter cake is dried in a vacuum oven at 40°C until ready for use, yielding NHS-PEG-NHS (number-average molecular weight 2282 Da).

[0083] Dissolve 20g PEG (0.01mol) in 60ml dichloromethane, add 2g triethylamine (0.02mol), and mix well; at the same time, dissolve 3.78g 4-(chloromethyl)benzoyl chloride (0.02mol) in 10ml dichloromethane, and inject the mixture in an ice bath for 5-10 min. oThe mixture was injected into the PEG solution at 40 °C and stirred at room temperature for 12 h. The resulting product was filtered and then rotary evaporated. The remaining solid was washed with deionized water and the supernatant was centrifuged and freeze-dried for later use to obtain Cl-PEG-Cl (number average molecular weight 2140 Da).

[0084] Dissolve 10 g of polylysine (0.002 mol, number-average molecular weight 5 kDa, same below) in 100 ml of isopropanol and stir at 60°C for 5 min. Add 5 g of NaOH to alkalize for 1 h. Then add 5 g of N,N-dimethylaminopropane chloride hydrochloride and reflux for 5 h. Filter the solution, wash the remaining solid twice with acetone, add deionized water, adjust the pH to 5 with acetic acid, dialyze against deionized water for 5 days, and lyophilize to obtain tertiary amine polylysine.

[0085] Dissolve 100 mg of NHS-PEG-NHS and 40 mg of 4-(chloromethyl)benzoyl chloride in 1 ml of 0.2 mol / L borate buffer (pH 9.8), designated as component A. Dissolve 150 mg of tertiary amine polylysine in 1 ml of borate buffer, designated as component B. Components A and B are separately placed in a double-barreled syringe and injected at the site of action to rapidly form a gel.

[0086] Comparative Example 1 Preparation of single network gel

[0087] Thoroughly dissolve 10g of PEG (0.005mol, number-average molecular weight 2000Da) in 15ml of dichloromethane. Simultaneously, dissolve 2.56g of DSC (0.01mol) in 15ml of acetonitrile by sonication for 15 minutes. Combine the two solutions, add 0.5g of DMAP (0.004mol), and sonicate again for 30 minutes. Stir the suspension at 25°C for 10 hours. Rotary evaporate the product, wash it three times with ether, and filter it. Dry the filter cake in a vacuum oven at 40°C to obtain NHS-PEG-NHS (number-average molecular weight 2282Da).

[0088] Dissolve 100 mg of NHS-PEG-NHS in 1 ml of PBS (sodium chloride concentration: 1.45 mmol / mL, sodium hydrogen phosphate: 0.09 mmol / mL, sodium dihydrogen phosphate: 0.005 mmol / mL, pH = 7.4), denoted as component A. Dissolve 100 mg of PEI in 1 ml of PBS, denoted as component B. Components A and B are separately placed in a double-barreled syringe and injected at the site of action to rapidly form a gel.

[0089] Comparative Example 2 Preparation of Single Network Gel

[0090] Thoroughly dissolve 20 g of PEG (0.005 mol, number-average molecular weight 4000 Da) in 30 ml of chloroform. Simultaneously, dissolve 2.56 g of DSC (0.01 mol) in 15 ml of acetonitrile by sonication for 15 minutes. Combine the two solutions, add 0.5 mL of pyridine (0.006 mol), and sonicate again for 30 minutes. Stir the suspension at 25°C for 10 hours. The product is then rotary evaporated, washed three times with ether, and filtered. The filter cake is dried in a vacuum oven at 40°C until ready for use, yielding NHS-PEG-NHS (number-average molecular weight 4282 Da).

[0091] Dissolve 5g of chitosan in 100ml of isopropanol and stir at 60°C for 5 minutes. Add 5g of NaOH to alkalize the mixture for 1 hour. Then add 5g of N,N-dimethylaminopropane chloride hydrochloride and reflux for 5 hours. Filter the mixture, wash the remaining solid twice with acetone, add deionized water, adjust the pH to 5 with acetic acid, dialyze against deionized water for 5 days, and lyophilize to obtain tertiary amine chitosan.

[0092] Dissolve 150 mg of NHS-PEG-NHS in 1 ml of PBS (85 mg of sodium chloride, 13.22 mg of disodium hydrogen phosphate, and 0.6 mg of sodium dihydrogen phosphate), designated as component A. Dissolve 100 mg of tertiary amine chitosan in 1 ml of PBS, designated as component B. Components A and B are separately placed in a double-barreled syringe and injected at the site of action to rapidly form a gel.

[0093] Comparative Example 3 Preparation of Single Network Gel

[0094] Thoroughly dissolve 2 g of PEG (0.005 mol, number-average molecular weight, 400 Da) in 15 ml of acetonitrile. Simultaneously, dissolve 3.68 g of DSS (0.01 mol) in 15 ml of acetonitrile by sonication for 30 minutes. Combine the two solutions, add 0.7 mL of triethylamine (0.005 mol), and sonicate again for 30 minutes. Stir the suspension at 25°C for 10 hours. Rotary evaporate the product, wash it three times with ether, and filter it. The filter cake is dried in a vacuum oven at 40°C until ready for use, yielding NHS-PEG-NHS (number-average molecular weight, 850 Da).

[0095] Dissolve 3g of chitosan in 100ml of isopropanol and stir at 60°C for 5 minutes. Add 5g of NaOH to alkalize the mixture for 1 hour. Then add 5g of N,N-dimethylaminopropane chloride hydrochloride and reflux for 5 hours. Filter the mixture, wash the remaining solid twice with acetone, add deionized water, adjust the pH to 5 with acetic acid, dialyze against deionized water for 5 days, and lyophilize to obtain tertiary amine chitosan.

[0096] Dissolve 150 mg of NHS-PEG-NHS in 1 ml of PBS (85 mg of sodium chloride, 13.22 mg of disodium hydrogen phosphate, and 0.6 mg of sodium dihydrogen phosphate), designated as component A. Dissolve 100 mg of tertiary amine chitosan in 1 ml of PBS, designated as component B. Components A and B are separately placed in a double-barreled syringe and injected at the site of action to rapidly form a gel.

[0097] Comparative Example 4 Preparation of Single Network Gel

[0098] Thoroughly dissolve 10 g of PEG (0.01 mol, number-average molecular weight 1000 Da) in 15 ml of acetonitrile. Simultaneously, dissolve 3.26 g of DSG (0.01 mol) in 15 ml of chloroform by sonication for 15 minutes. Combine the two solutions, add 0.5 mL of pyridine (0.006 mol), and sonicate again for 30 minutes. Stir the suspension at 25°C for 10 hours. Rotary evaporate the product, wash it three times with ether, and filter it. Dry the filter cake in a vacuum oven at 40°C to obtain NHS-PEG-NHS (number-average molecular weight 1270 Da).

[0099] Dissolve 100 mg of NHS-PEG-NHS in 1 ml of PBS (85 mg of sodium chloride, 13.22 mg of disodium hydrogen phosphate, and 0.6 mg of sodium dihydrogen phosphate), referred to as component A. Dissolve 80 mg of PEI in 1 ml of PBS, referred to as component B. Each component, A, is separately placed in a double-barreled syringe and injected into the target site to rapidly form a gel.

[0100] Comparative Example 5 Preparation of Single Network Gel

[0101] Thoroughly dissolve 10 g of PEG (0.005 mol, number-average molecular weight, 2000 Da) in 15 ml of dichloromethane. Simultaneously, dissolve 2.56 g of DSC (0.01 mol) in 15 ml of acetonitrile by sonication for 15 minutes. Combine the two solutions, add 0.5 g of DMAP (0.004 mol), and sonicate again for 30 minutes. Stir the suspension at 25°C for 10 hours. Rotary evaporate the product, wash it three times with ether, and filter it. The filter cake is dried in a vacuum oven at 40°C until ready for use, yielding NHS-PEG-NHS (number-average molecular weight, 2282 Da).

[0102] Dissolve 100 mg of NHS-PEG-NHS in 1 ml of water, labeled as component A; dissolve 100 mg of gelatin in 1 ml of water, labeled as component B. Fill components A and B separately into a double-barreled syringe and inject at the site of action to quickly form a gel.

[0103] Comparative Example 6 Preparation of Single Network Gel

[0104] Thoroughly dissolve 10 g of PEG (0.005 mol, number-average molecular weight 2000 Da) in 15 ml of dichloromethane. Simultaneously, sonicate 2.56 g of DSC (0.01 mol) in 15 ml of acetonitrile for 15 minutes. Combine the two solutions, add 0.5 g of DMAP (0.004 mol), and sonicate again for 30 minutes. Stir the suspension at 25°C for 10 hours. Rotary evaporate the product, wash it three times with ether, and filter it. The filter cake is dried in a vacuum oven at 40°C until ready for use, yielding NHS-PEG-NHS (number-average molecular weight 2282 Da).

[0105] Dissolve 10g of polylysine (number-average molecular weight, 5kDa, the same below) in 100ml of isopropanol and stir at 60°C for 5 minutes. Add 5g of NaOH to alkalize for 1 hour, then add 5g of N,N-dimethylaminopropane chloride hydrochloride and reflux for 5 hours. Filter the solution, wash the remaining solid twice with acetone, add deionized water, adjust the pH to 5 with acetic acid, dialyze against deionized water for 5 days, and lyophilize to obtain tertiary amine polylysine.

[0106] Dissolve 100 mg of NHS-PEG-NHS in 1 ml of 0.2 mol / L borate buffer (pH 9.8), designated as component A. Dissolve 150 mg of tertiary amine polylysine in 1 ml of borate buffer, designated as component B. Fill components A and B separately into a double-barreled syringe and inject at the site of action to rapidly form a gel.

[0107] Comparative Example 7

[0108] Thoroughly dissolve 10g of PEG (0.005mol, number-average molecular weight 2000Da) in 15ml of dichloromethane. Simultaneously, dissolve 2.56g of DSC (0.01mol) in 15ml of acetonitrile by sonication for 15 minutes. Combine the two solutions, add 0.5g of DMAP (0.004mol), and sonicate again for 30 minutes. Stir the suspension at 25°C for 10 hours. Rotary evaporate the product, wash it three times with ether, and filter it. Dry the filter cake in a vacuum oven at 40°C to obtain NHS-PEG-NHS (number-average molecular weight 2282Da).

[0109] Dissolve 27.5 g PEG (0.0025 mol, number average molecular weight 11000 Da) in 120 ml of dichloromethane, add 0.5 g triethylamine (0.0005 mol), and mix well. At the same time, dissolve 0.945 g 4-(chloromethyl)benzoyl chloride (0.0005 mol) in 10 ml of dichloromethane, and add the solution through a syringe in an ice bath for 5 to 10 minutes. oThe mixture was injected into the PEG solution at 40 °C and stirred at room temperature for 12 h. The resulting product was filtered and then rotary evaporated. The remaining solid was washed with deionized water and the supernatant was centrifuged and freeze-dried for later use to obtain Cl-PEG-Cl (number average molecular weight 11,000 Da).

[0110] Dissolve 100 mg of NHS-PEG-NHS and 60 mg of Cl-PEG-Cl in 1 ml of PBS solution (1.45 mmol / mL sodium chloride, 0.09 mmol / mL sodium hydrogen phosphate, 0.005 mmol / mL sodium dihydrogen phosphate, pH 7.4), referred to as component A. Dissolve 100 mg of PEI (number-average molecular weight 10 kDa, the same below), referred to as component B, in 1 ml of PBS solution. Components A and B were separately placed into double-barreled syringes and injected into the site of action.

[0111] like Figure 2 As shown in the figure, the addition of Cl-PEG-Cl with too high molecular weight not only prevents the second network from forming gel, but also affects the gelation of the first network.

[0112] Example 7 Self-reinforcement performance test of injectable gel

[0113] The compression tests of the hydrogels in Examples 1, 2, 6 and Comparative Examples 1, 2, 6 were conducted using a high and low temperature dual column testing machine. Figure 3 a shows a comparison of the compressive strength of the gels of Example 1 and Comparative Example 1, wherein the strength of the comparative hydrogel at 48 h was < 60 kPa, while the hydrogel of Example 1 reached 345.0 kPa. In addition, the modulus of the hydrogel of Comparative Example 1 remained almost unchanged with the extension of the reaction time, ultimately reaching ~33.6 kPa. The compressive modulus of the hydrogel of Example 1 gradually increased, ultimately reaching ~70.4 kPa. The modulus of the gel continued to rise within 48 h after injection, proving that the strength of the hydrogel slowly increased within a certain period of time after injection. Similarly, Figure 3 b. The tertiary amine chitosan system can also prove that the compression strength of the double network hydrogel increases over time. This is because after the hydrogel quickly reacts with amide to form a gel, the slow reaction between the tertiary amine group and the halogen element group continues to increase the strength. This slow enhancement feature not only enhances the strength of the injectable hydrogel itself, but also enables it to better cover wounds in daily environments. It is also more flexible in wound treatment and can achieve the purpose of slowly adapting the wound to the coverage and filling of the hydrogel after injection, thus preventing secondary damage to the wound and damage to surrounding tissues. In addition, Figure 3 Although the double network gel given in c has a tendency to strengthen over time compared with the single network hydrogel, the strength after strengthening is far less than that of the single network hydrogel. Figure 3 a, 3b, this is because the network structure formed after the short-chain small molecules are added as the second component is not as good as that of the synthetic polymer.

[0114] Example 8 Antibacterial Performance Test of Injectable Gel

[0115] The antibacterial properties of the gels of Comparative Example 1 and Example 1 were tested with Escherichia coli (E. coli, Gram-negative) and Staphylococcus aureus (S. aureus, Gram-positive). Single colonies of E. coli and Staphylococcus aureus cultured on LB solid medium for 12 hours were picked and inoculated into LB liquid medium at 37 o C, shaken at 160 rpm overnight. The bacterial solution was diluted to a standard concentration (the absorbance at 600 nm was 0.1 for E. coli and 0.05 for S. aureus). The hydrogel was placed in a well plate and the standard concentration of bacterial solution was injected. Fluorescence microscopy was used to observe the staining 24 hours (E. coli) and 12 hours (S. aureus). The results are shown in Figure 2. Figure 4 As shown in Figure 2, after a certain period of incubation, the gel of Example 1 containing a cross-linked network of amino groups and succinimide and a cross-linked network of halogen elements and tertiary amines has better antibacterial performance than the gel of Comparative Example 1 containing only an aminosuccinimide network due to the formation of antibacterial quaternary ammonium groups. The sterilization rates of the gels of all the examples and the comparative examples were statistically analyzed as follows: Figure 5 The sterilization rates of all the examples are better than those of the corresponding comparative examples, and the sterilization rates for the two bacteria are around 80%.

Claims

1. A biodegradable hydrogel, characterized in that The biodegradable hydrogel is made of component A and component B: The component A is composed of the following components: NHS-PEG-NHS, halide-PEG-halide, and solvent A; solvent A is water or saline solution; The number average molecular weight of NHS-PEG-NHS is 400~5000Da; the number average molecular weight of halide-PEG-halide is 400~5000Da; The component B is composed of the following components: a macromolecular substance containing an amino group, and a solvent B; the solvent B is water or a saline solution; The amino-containing macromolecular substance is one or a mixture of two or more of tertiary amine chitosan, tertiary amine polylysine, gelatin, and polyethyleneimine, and the number average molecular weight of the amino-containing macromolecular substance is 5 to 100 kDa; The mass ratio of the NHS-PEG-NHS, halide-PEG-halide and amino-containing macromolecular substance is 100:20-90:15-180; the mass of the NHS-PEG-NHS is 25-100 mg / mL based on the total volume of component A and component B; The structure of the NHS-PEG-NHS is one of the following: II III In formula III, x is an integer between 1 and 10; The structure of the halide-PEG-halide is shown below: I In formula I, R1 is a halogenated C1-C6 alkyl group.

2. The biodegradable hydrogel according to claim 1, wherein: The solvent A and the solvent B are each independently one of water, PBS, borate buffer solution, and physiological saline, or a mixture of two or more solvents.

3. The biodegradable hydrogel according to claim 1, wherein: Tertiary amine chitosan and tertiary amine polylysine are prepared according to the following method: chitosan or polylysine is dissolved in an organic solvent, an alkaline substance is added for alkalization for 1 hour, an amine compound is added and refluxed at room temperature for 5 hours, and the resulting reaction solution is post-treated to obtain the tertiary amine chitosan or tertiary amine polylysine; the amine compound is one or a mixture of N,N-dimethylaminochloropropane hydrochloride and N,N-dimethyl-oxiranylmethylamine.

4. The biodegradable hydrogel according to claim 3, wherein: The post-treatment comprises: filtering the reaction solution, washing the filter cake with acetone, adding deionized water, adjusting the pH to 5 with acetic acid, dialyzing with deionized water for 5 days, and freeze-drying the retentate to obtain the tertiary amine chitosan or tertiary amine polylysine; The molar ratio of the chitosan or polylysine to the amine compound is 1:15 to 100; The organic solvent is one or a mixture of two or more of isopropyl alcohol, dichloromethane and chloroform; The alkaline substance is one or a mixture of two or more of NaOH, NaHCO3, and Na2CO3, and the mass ratio of the alkaline substance to the amino macromolecule is 1:1-2; The volume of the organic solvent is 10-20 ml / g based on the mass of the chitosan or polylysine.

5. The method for preparing the biodegradable hydrogel according to claim 1, wherein The method comprises the following steps: filling the component A and the component B into different barrels of a double-barrel syringe respectively, mixing and extruding the mixture, and obtaining the biodegradable hydrogel.

Images